Parathyroid hormone (PTH) is produced in the parathyroid, and plays an important role, acting on the bone and the kidney which are its target organs to control the blood calcium and phosphate ion levels. PTH is a peptide hormone composed of 84 amino acids, and its biological activity is known to be able to be reproduced by the N-terminal (the 1 to 34-positions) peptide fragment [G. W. Tregear et al., Endocrinology, 93, 1349-1353 (1973)].
This N-terminal (the 1 to 34-positions) peptide fragment of human PTH (hereinafter briefly referred to as "human PTH(1-34)") has the following amino acid sequence: EQU 1 2 3 4 5 6 7 8 9 10 11 12 H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-13 14 15 16 17 18 19 20 21- 22 23 24 25 26 Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-27 28 29 30 31 32 33 34 Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH (SEQ ID NO: 1)
In order to understand the structure-activity relationship of said hormone, various derivatives of the PTH(1-34) fragment have been synthesized. Previously, investigations of bovine PTH(1-34) have been mainly conducted. However, recent investigations are increasingly directed to human PTH(1-34). For example, conversion of the C-terminal Phe of human PTH(1-34) to Phe-NH.sub.2 is known to cause a rise in activity [JP-A-58-96052 (the term "JP-A" as used herein means an "unexamined published Japanese patent application")]. However, this is considered that decomposition caused by carboxypeptidase is inhibited, resulting in an apparent rise in activity. For a molecule in which 2 Met residues contained in human PTH(1-34) are substituted by Nle residues, hormone activity is known to be prevented from disappearance by oxidation (JP-A-61-24598).
F. E. Cohen et al. [The Journal of Biological Chemistry, 226, 1997-2004 (1991); WO 92/00753] substituted various L-amino acids for Ser at the 3-position in human PTH(1-34) and bovine PTH(1-34). As a result, Ala-substituted derivatives showed activity approximately equivalent to that of the natural type fragments, but derivatives substituted by the other amino acids are extremely lowered in activity. Further, substitution of amino acids at the 6- and 9-positions does not provide derivatives having activity suitable for use as medical drugs. Furthermore, Wo 93/06845 discloses that even when the sequence of the consecutive basic amino acids of the 25- to 27-positions of PTH(1-34) is substituted by another amino acid sequence, its biological activity is retained, but activity on blood pressure or on smooth muscle is decreased. WO 93/06846 also discloses that an analogue in which the 23-position is substituted by another amino acid has a similar effect. In addition, JP-A-6-184198 (WO 94/02510) discloses various analogues substituted by amino acid, as well as analogues in which amino groups of side chains are modified.
From biological activity of PTH, it is expected that PTH can be used as drugs useful for various bone diseases, etc. However, the following properties of the peptide make this difficult.
(1) PTH is easily decomposed by various enzymes in the body;
(2) The absorption efficiency of PTH into the body by various routes is very low; and
(3) PTH is unstable under various physical and chemical conditions such as oxidation.
In order to solve such problems, and to elucidate the structure-activity relationship of said hormone, various derivatives of the PTH(1-34) active fragment have been synthesized. On measurement of biological activity of these compounds, compounds avoiding any of the problems of the above (1) to (3) have enhanced activity in some cases as described above with respect to the derivative having Phe-NH.sub.2 at the 34-position. Derivatives enhanced in inherent activity, for example, by an increase in affinity or receptors can compensate for the problems of the above (1) to (3) by their high activity.
Previously, the present inventors made substitution of amino acids of human PTH(1-34) by chemical synthesis and have discovered that this object were attained by (1) subjecting any of the amino acids at the 1-, 8-, 11-, 12-, 13-, 18-, 19-, 21- 23-, 25-, 26-, 27- and 34-positions of human PTH(1-34) to amino acid substitution considering the resistance to various proteases, (2) enhancing activity of said hormone by amino acid substitution considering two-dimensional structure to be expected, hydrophilicity, hydrophobicity or ionic environment, or (3) substituting amino acids unstable to acidic or alkaline conditions, oxidation conditions, etc. by amino acids stable to these conditions without reducing activity, and have provided excellent human PTH(1-34) derivatives (JP-A-5-32696). Further, the present inventors discovered that derivatives of said peptide obtained by substitution of any of the amino acids at the 3-, 14-, 15-, 16-, 17-, 25-, 26-, 27- and 34-positions of the human PTH(1-34) sequence, or a combination thereof have excellent activity (JP-A-5-320193).
Furthermore, the present inventors discovered that a peptide derivative in which any of the amino acids at the 34- to 47-positions of human PTH(1-84) is substituted by Cys can form a dimer, and that introduction of another functional group can convert the peptide to a compound having more desirable properties (JP-A-5-271279).